A total of 30 patients aged 25–35 years (mean age: 31) with moderate to severe IUA underwent IUA dissection in the Affiliated Yantai Yuhuangding Hospital of Qingdao University (Shandong, China) between January 2016 and December 2016 and were enrolled in the present study. The criteria for patient inclusion were as follows: Initial hysteroscopic adhesions score in line with moderate to severe IUA [American Fertility Society score (12) ≥5]; age ≤40 years; hysteroscopic examination following surgery; and a complete record of the medical history. A total of 30 females aged 23–34 years (mean age: 28) with normal endometrium who received hysteroscopic examination were selected as controls. Endometrial tissue was collected by curettage. All participants provided written informed consent and the study was approved by the Ethics Committee of The Affiliated Yantai Yuhuangding Hospital of Qingdao University.

Expression levels of EGF and PDGF-BB in endometrial tissue were detected using RT-qPCR. Additionally, expression levels of epithelial membrane antigen (EMA), Thy-1 membrane glycoprotein (THY-1), cytokeratin (CK), collagen type 1 (Col I), integrin α-6 (CD49f), vimentin and 5B5 in ESCs were assessed using RT-qPCR. Total RNA was extracted from endometrial tissue and ESCs using TRIzol reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The concentration and quality of RNA were assessed using the 260/280 nm absorbance ratio of 1.8–2.0. Total RNA was reverse transcribed using a reverse transcription kit (Takara Bio, Inc., DRR047A) according to the manufacturer's protocol. RT-qPCR was performed using LightCycler^®480 SYBR Green Master mix (Roche Diagnostics, Basel, Switzerland). Primers used for the PCR are listed in Table I. All primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). GAPDH was used as an endogenous control. Thermocycling conditions were as follows: 95°C for 30 sec, followed by 40 cycles of 95°C for 10 sec, 60°C for 15 sec and 72°C for 15 sec, and finally, 60°C for 15 sec. Relative expression levels were determined using the 2^−ΔΔCq method (13).

Total protein was extracted from the endometrial tissues and EnSCs, and quantified using a bicinchoninic acid assay (Pierce; Thermo Fisher Scientific, Inc.). A total of 30 µg protein/lane was separated by SDS-PAGE (10% gels) and transferred onto polyvinylidene difluoride membranes. The membranes were then blocked with 5% non-fat milk. for 1 h at room temperature. Following blocking, the membranes were incubated with the following primary antibodies: Rabbit anti-human EGF antibody (cat. no. DF2225, 1:2,000; Affinity Biosciences, Cincinnati, OH, USA), rabbit anti-human PDGF-BB (cat. no. orb303833, 1:2,000; Biorbyt, Cambridge, UK,), rabbit anti-human EMA antibody (cat. no. orb31710; 1:2,000; Biorbyt), rabbit anti-human CK antibody (cat. no. BF0141, 1:1,000; Affinity Biosciences), rabbit anti-human CD49f antibody (cat. no. orb39949, 1:1,000; Biorbyt), rabbit anti-human THY-1 (cat. no. orb136416, 1:2,000; Biorbyt), rabbit anti-human Col I antibody (cat. no. orb345868, 1:2,000; Biorbyt), rabbit anti-human 5B5 antibody (cat. no. orb241612, 1:1,000; Biorbyt), rabbit anti-human vimentin antibody (cat. no. AF7013, 1:2,000; Affinity Biosciences), rabbit anti-rat estrogen receptor (ESR1) (cat. no. DF6094, 1:2,000; Affinity Biosciences) and rabbit anti-rat matrix metalloproteinase (MPP)-9 (cat. no. orb100446, 1:1,000; Biorbyt) overnight at 4°C. Membranes were washed three times with Tris-buffered-saline with Tween-20 (TBST) for 10 min. Following the primary incubation, membranes were incubated with horseradish peroxidase-labeled goat anti-rabbit antibody (cat. no. orb345943, 1:1,500; Biorbyt) at room temperature for 1 h. Membranes were then washed three times with TBST. Protein bands were visualized using enhanced chemiluminescence (ECL; Merck KGaA). The densitometric analysis for the quantification of the bands was performed using ImageJ software (version 2.1; National Institutes of Health, Bethesda, MD, USA). β-actin was used as an endogenous control.

Endometrial tissue was obtained from ovulating women without endometrial disease. All participants provided written informed consent and the study was approved by the Ethics Committee of The Affiliated Yantai Yuhuangding Hospital of Qingdao University. EnSCs were prepared as reported by Ebrahimi-Barough et al (14,15). Endometrial tissue was rinsed with PBS and cut into 1 mm³ pieces. Tissues were then incubated with 5 ml digestion solution in a 25 cm² cell culture flask [0.25% trypsin (cat. no. T8150; Beijing Solarbio Science & Technology Co, Ltd., Beijing, China) + 0.02% ethylenediaminetetraacetic acid (cat. no. C1033; Beijing Solarbio Science & Technology Co., Ltd.) + 300 µg/ml type II collagenase (cat. no. C8150; Beijing Solarbio Science and Technology Co., Ltd.)] under agitation (80 rpm) for 45–60 min at 37°C. Samples were filtered with 80 mesh screen and collect the filtrate. Red blood cells were removed with red blood cell lysis buffer (cat. no. ab204733; Abcam). When the cells were ~80% confluent, they were used for subsequent experiments.

EnSCs were fixed with 4% paraformaldehyde at room temperature for 20 min and permeabilised with 0.1% Triton in PBS at room temperature for 20 min. The slides were then blocked using 5% goat serum in TBS and incubated with primary antibody against CD90 (cat. no. ab106934, 1:200; Abcam) overnight at 4°C. Following the primary incubation, cells were incubated with horseradish peroxidase secondary antibody (cat. no. ab95376; rabbit anti-rat immunoglobulin; 1:200; Abcam) at 37°C for 4 h. Cells were washed with TBST for 10 min. The negative controls were incubated only with the secondary antibody. The slides were stained with 3,3-diaminobenzidine tetrahydrochloride for 10 min, counterstained with hematoxylin, subjected to gradient alcohol dehydration and mounted with neutral gum. Cells were observed under an optical microscope (×400 magnification).

A total of 50 specific pathogen-free (SPF) female Sprague Dawley rats (weight, 200–220 g, 8 weeks old) were purchased from the Shanghai Institutes for Biological Sciences, Chinese Academy of Science (Shanghai, China). The animals were maintained in a 12–12 h light-dark cycle at a temperature of 22±1°C with free access to food and water in a specific pathogen-free environment. A total of 50 rats were randomly divided into 5 groups with 10 rats in each group. A rat model of IUA was established by scraping the uterine horn to mimic the cause of IUA in humans. The model was established using the methods described by Tang et al (16). Rats in the control group were subjected to a sham surgery. At day 7 following the establishment of the model, rats in the EnSCs group were subcutaneously injected with 1 ml EnSCs, rats in estrogen group were subjected to subcutaneous injection of estradiol benzoate (0.5 mg/kg; Hangzhou Animal Pharmaceutical Factory, China) in PBS according to previous studies (17) and rats in the E+EnSCs group received an injection of estradiol benzoate (0.5 mg/kg) and EnSCs (1 ml). Rats (n=5/group) were sacrificed 1 week after IUA and 5 weeks following IUA. Uterine tissue was collected from both sides of the uterus and 2 ml of blood was extracted. Blood samples were centrifuged at 3,000 × g at 4°C for 10 min. Serum was stored at −80°C. The study was approved by the Animal Ethics Committee of The Affiliated Yantai Yuhuangding Hospital of Qingdao University.

Uterine tissue was fixed in 10% neutral formaldehyde and embedded in paraffin. Paraffin-embedded tissue samples were cut into 5-µm sections. The tissue sections were deparaffinized in xylene at room tempetature and rehydrated in a descending ethanol series (100% for 5 min, 95% for 1 min, 80% for 5 min and 75% for 5 min). Hematoxylin and eosin staining was performed using the routine method (18). Following dehydration, sections were treated with xylene for 2 times. Tissue sections were mounted with neutral resin and observed under optical microscopy (×200 magnification) (Olympus BX51; Olympus Corporation, Tokyo, Japan).

The total protein extracted from the blood samples of the experimental rat groups was used to determin the serum concentrations of β-estradiol (E2) (cat. no. KGE014), TGF-β1 (cat. no. MB100B), EGF (cat. no. DY3214) and PDGF-BB (cat. no. MBB00), as detected using ELISA kits (R&D Systems, Inc., Minneapolis, MN, USA), according the manufacturer's protocol.

Data were analyzed using SPSS software (version 19.0; IBM Corp., Armonk, NY, USA). The relevant data are expressed as the mean ± standard deviation. Results were analyzed using Student's t-test when only two groups were compared. Multiple comparisons between more than two groups were performed by one way analysis of variance followed by least significant difference or Kruskal-Wallis tests. P<0.05 was considered to indicate a statistically significant difference.

As presented in Fig. 1A and B, mRNA and protein levels of EGF and PDGF-BB were significantly increased in endometrial tissue derived from patients with IUA compared with that in healthy individuals (P<0.05). These results suggest that endometrial injury may significantly increase the expression levels of EGF and PDGF-BB.

As presented in Fig. 2, expression levels of endometrial epithelial stem cell-associated genes, including CK, EMA and CD49f, and endometrial stromal cell-associated genes, including THY-1 (CD90), Col I, 5B5 and vimentin, were significantly increased in EnSCs compared with that in the controls (P<0.05).

Isolated EnSCs were positive for CD90, as assessed using immunocytochemistry (Fig. 3A and B).

Hematoxylin and eosin staining demonstrated that the uterine cavity of the control group was covered by columnar epithelium and oval glands. In the model group, the uterine cavity was covered with flat columnar epithelial cells or stratified flat epithelial cells, and glands were rare. The number of endometrial glands in the estrogen, EnSCs and E+EnSCs groups was increased, and the newly formed glands were round or oval. No significant difference in the number of glands was detected between the EnSCs and estrogen groups. Newly formed glands were detected in the E+EnSCs group (as indicated by the black arrows; Fig. 4). These results suggest that treatment efficacy in the E+hESCs group may be improved compared with that in the remaining groups.

As presented in Fig. 5A, no significant difference in the expression of E2 was detected 1 week after IUA. The results demonstrated that the levels of serum E2 in the estrogen, EnSCs and E+EnSCs groups were significantly higher than that in the control group (P<0.05). However, no significant differences were detected between the model and control groups. Additionally, the expression of E2 was significantly increased in the estrogen and E+EnSCs groups compared with that in the model group (P<0.05). Additionally, a significant decrease in the E2 content was detected in the EnSCs and estrogen groups compared with that in the E+EnSCs group (P<0.05; Fig. 5A).

One week after IUA, the levels of EGF, TGF-β1 and PDGF-BB were significantly increased in the model, EnSCs, estrogen and E+EnSCs groups compared with those in the control group (P<0.05; Fig. 5B-D). The levels of TGF-β1, EGF and PDGF-BB were significantly decreased in the EnSCs, estrogen and E+EnSCs groups compared with those in the model group at 4 weeks post-treatment (P<0.05; Fig. 5B-D). No significant difference in the expression of EGF, TGF-β1 and PDGF-BB was detected between the EnSCs and estrogen groups (Fig. 5B-D). Additionally, a significant decrease in the levels of TGF-β1, EGF and PDGF-BB was detected in the E+EnSCs group compared with that in the EnSCs and estrogen groups (P<0.05; Fig. 5B-D).

One week after IUA, the expression levels of ESR1 and MMP-9 were significantly decreased in the model, EnSCs, estrogen and E+EnSCs groups compared with those in the control group (P<0.05; Fig. 6A-C), as assessed using western blot analysis. The expression levels of ESR1 and MMP-9 were significantly increased in the estrogen, EnSCs and E+EnSCs groups compared with those in model group at 5 weeks after IUA (P<0.05; Fig. 6B and C). No significant difference in expression levels of ESR1 and MMP-9 were detected between the estrogen and EnSCs groups at 5 weeks after IUA. However, the expression levels of ESR1 and MMP-9 were significantly decreased in the estrogen and EnSCs groups compared with those in the E+EnSCs group (P<0.05; Fig. 6B and C).